The overall goal is to begin to identify and define the molecular basis of cell-mediated cytolysis. Murine allogeneic cytotoxic T lymphocytes (CTL) with high levels of activity will be used as the model system in these studies. These CTL can be readily generated in mixed lymphocyte cultures using responder cells from immunized mice and further expanded by culture in medium with T cell growth factor activity. Enriched CTL populations that are actively growing will be used in two general approaches to define the molecular bases of CTL functions. The first approach will be to identify the molecule(s) that is susceptible to specific types of inhibitors of CTL-mediated lysis (CML). For example, we have found that thiol-reactive compounds and substrates and inhibitors of serine-dependent proteases block CML (Redelman and Hudig, J. Immunol. 124, p. 870, 1980). More recently we have confirmed that inhibitors of methylation also inhibit CML. CTL will be labelled intrinsically or surface iodinated and then reacted with a nonpenetrating thiol specific hapten or with a macromolecular antiprotease that binds firmly to serine-dependent proteases. The relevant molecules will then be immunoprecipitated from cell lysates using antibody to the thiol-reactive hapten or to the antiprotease. Other studies will isolate the putative protease directly by preparing and using a monoclonal antibody to the active site of serine-dependent proteases. The molecule(s) methylated during CML will be labelled by using a radioactive methyl donor. Immunoprecipitated or methylated molecules will be analyzed by polyacrylamide gel electrophoresis or by chromatography if methylated phospholipids are implicated. The second approach to relative structure and function will be to attempt to define the function of molecules reactive with anti-effector cell antibodies that block CML. Most antieffector antibodies do not inhibit CML, although a few such antibodies have been described (e.g., Redelman and Trefts, J. Immunol. 121, p. 1532, 1979). Conventional antithymocyte and monoclonal anti-T200 antibodies, both of which block CML, will be examined to define the molecules with which they react and/or to determine the phase(s) of CML that is blocked. It will also be determined whether these inhibitory antibodies react with any of the molecules identified in the preceding experiments examining thiols, proteases, and methylation products. (CS)